Inflammatory Diseases and the Role of n-7 Unsaturated Fatty Acids as Functional Lipids

Abstract

With the increasing childbearing age, the number of mothers with diabetes and gestational diabetes is escalating. Maternal hyperglycemia creates an intrauterine hyperglycemic environment via the placenta, which causes signaling abnormalities in various fetal organs due to excessive glycation. This is associated with future disease development in the child. We have shown that insulin signaling defects are induced in fetal cardiomyoblasts using a rat gestational diabetes mellitus model and cellular models. Furthermore, we reported that maternal intake of eicosapentaenoic acid (EPA), an n-3 unsaturated fatty acid, during pregnancy can ameliorate this signaling defect. However, EPA has anti-coagulant effects, and the pollution of marine fish oil, the source for EPA supplements, raises concerns about active intake by pregnant women. Recently, palmitoleic acid, an n-7 unsaturated fatty acid, garnered attention as a candidate functional lipid alternative to EPA because it has been reported to have anti-obesity, lipid metabolism improvement, and cardioprotective effects similar to those of EPA. Palmitoleic acid has cis and trans structural isomers, which differ in their food intake route and metabolism in humans. This article introduces recent findings on the biological functions of palmitoleic acid in lifestyle-related diseases and cardiovascular diseases, ranging from basic research to clinical studies.

1. Introduction

Prenatal nutrition has a significant impact on the long-term health of the unborn child. In particular, the cardiovascular epidemiological studies by Barker et al. have shown that the intrauterine environment during pregnancy is closely related to the development of future lifestyle-related diseases [1, 2, 3, 4, 5]. Gluckman and Hanson proposed the Developmental Origins of Health and Disease hypothesis, which states that predisposition to lifestyle-related diseases is shaped by gene-environment interactions during fertilization, embryonic development, fetal life, and infancy, and that mismatches with the fetal environment after birth lead to the development of diabetes and hypertension [6, 7]. Many of these studies have suggested that a low-nutrition environment in the womb during the fetal period increases the likelihood that the child will later suffer from lifestyle-related diseases, such as obesity, diabetes, and hypertension in the future [8, 9, 10].

In the post-World War II period, economically developed countries entered an era of global food satiation. Economic growth also changed people’s lifestyles. These social conditions have been accompanied by an increase in late marriages, an increase in maternal obesity, an increase in maternal age, an increase in the incidence of gestational diabetes mellitus (GDM), and an increase in pregnant women with type 2 diabetes. The nutritional environment of pregnant women has increasingly become overnourished rather than undernourished [11, 12, 13]. The fetus of a pregnant woman with such an abnormal glucose metabolism is exposed to an intrauterine hyperglycemic environment, through the umbilical cord, due to maternal hyperglycemia. As a result, neonatal complications, such as gigantism, hypoxia, respiratory disorders, congenital malformations, and myocardial hypertrophy, have been reported in children born to diabetic pregnant women [14, 15, 16, 17, 18]. However, the molecular mechanisms by which this intrauterine hyperglycemic environment during the fetal period contributes to the future health and disease development of the child after birth are not well understood. We are conducting basic research using animal and cellular models to elucidate the underlying molecular mechanisms and to search for foods that show diabetes-preventive effects in pregnant women. In this chapter, we present recent findings on functional lipids that may prevent cardiac disease in children born to diabetic mothers.

2. Fetal heart in an intrauterine hyperglycemic environment

We created a GDM model rat by administering streptozotocin into the tail vein of Wistar rats on gestation day 2. To determine the effects of a high-fat diet during pregnancy on the infants, we fed the GDM model rats a high-fat lard diet containing saturated fatty acids and a control diet [19]. The stillbirth rate of GDM model rats fed a high-fat lard diet was significantly higher than that of GDM model rats fed a control diet [19]. Palmitic acid loading in lard has been reported to cause inflammation and cardiomyocyte dysfunction in animal- and cellular-level experiments [20, 21, 22, 23, 24, 25]. The GDM rat study suggested that not only was the fetus exposed to intrauterine hyperglycemia, but that the mother’s intake of palmitic acid from a high-fat lard diet during gestation may have further impaired cardiac function in the offspring [19].

Based on reports that fish oil has a positive effect on cardiovascular disease [26, 27], we fed GDM model rats a high-fat fish oil diet, a high-fat lard diet, and a control diet and analyzed the nutrient signals from the hearts of the infants. We found that the lard-fed GDM model rat offspring had decreased phosphorylation levels of Akt, which is important for sugar uptake [28]. However, when mothers were fed fish oil during pregnancy, the Akt phosphorylation level was increased, and insulin signaling was improved in the hearts of GDM model rat offspring [28]. The biological functional components in fish oil may have ameliorated the signaling impairment caused by hyperglycemia and palmitic acid loading.

We isolated primary cardiomyoblasts from the hearts of rat infants and established a cellular model of a hyperglycemic environment by elevating the glucose concentration in the medium to address two questions: Why was insulin signaling abnormal in the hearts of GDM model rat offspring, and what components of fish oil ameliorated this signaling abnormality? We focused on eicosapentaenoic acid (C20:5n-3: EPA), which is abundant in fish oil and has known cardiovascular protective effects [29, 30, 31, 32, 33]. We orally administered EPA to GDM model rats during gestation and analyzed neonatal primary cardiomyoblasts isolated from the offspring. The results showed that insulin signaling was inhibited in primary cardiomyoblasts of GDM model rat offspring and that insulin resistance had been induced in these rat infants [34].

However, insulin resistance was improved in the hearts of infants born to GDM model rat mothers that were orally administered EPA [34]. Furthermore, in primary cardiomyocytes from neonatal rats exposed to an intrauterine hyperglycemic environment, intracellular reactive oxygen species (ROS) were chronically elevated, and excess advanced glycation end-products (AGEs) were observed, as compared with normal cells. AGE formation has recently attracted attention as a cause of aging and disease [35, 36, 37, 38, 39, 40]. Furthermore, AGEs increase the expression of receptors for AGEs (RAGEs) and induce AGE-RAGE signaling. This signaling also increases the expression of genes encoding various inflammatory cytokines (IL-6, TNFα, NF-κB) via phosphorylation of Jun amino-terminal kinase (JNK).

These results indicated that the fetuses of GDM model rats are exposed to a hyperglycemic environment in utero, resulting in chronic inflammation by accumulating AGEs. However, feeding EPA to pregnant GDM model rats suppressed the production of AGEs and intracellular ROS in the offspring, resulting in the amelioration of signaling abnormalities [34]. Therefore, it is highly likely that EPA was responsible for the improvement of cardiac insulin signaling. In that regard, a summary is shown in Figure 1.

Figure 1.

Intrauterine hyperglycemia environment causes impaired signaling in the fetal heart. In gestational diabetes mellitus, maternal hyperglycemia creates a hyperglycemic intrauterine environment via the placenta. In this environment, advanced glycation end-products (AGEs) of proteins accumulate in fetal cardiomyoblasts, triggering inflammatory signals and impairing nutrient signaling by reactive oxygen species in the cells. Maternal intake of eicosapentaenoic acid (EPA), present in fish oil, during fetal life improves signaling by inhibiting formation of AGEs.

3. n-3 unsaturated fatty acid intake issues for pregnant women

Our experiments with the GDM rat model revealed that EPA improves the intrauterine hyperglycemic environment during pregnancy. EPA is one of the fatty acids transported from the mother to the fetus via the placenta [41, 42] and is a very important essential fatty acid for the fetus [43, 44, 45]. However, EPA is known to have anticoagulant and thromboprophylaxis effects [46, 47, 48, 49]. Therefore, very high intakes or levels of EPA may cause health problems, such as hemorrhage, prolonged gestation, or premature delivery, and thus intakes in pregnant women are being examined from various perspectives [50, 51]. In addition, given marine pollution in many areas, EPA and DHA extracted from fish oil have been found to be contaminated with environmental contaminants, such as methylmercury and polychlorinated biphenyls, which are known to cause neurological disorders when ingested by pregnant women and which can adversely affect the fetus [45, 50, 51, 52, 53, 54, 55, 56]. Therefore, it is recommended that EPA derived from marine products with high methylmercury concentrations, such as tuna and swordfish, be avoided.

4. Palmitoleic acid as n-7 unsaturated fatty acid

Our research using GDM model rats revealed that intrauterine hyperglycemia causes chronic inflammation in cardiomyocytes and various organs during fetal development due to the oxidative stress caused by excessive AGEs and that this causes disease development in the offspring. It was also found that maternal intake of EPA, an n-3 unsaturated fatty acid, during the fetal period ameliorates this chronic inflammation. However, given the concerns about the intake of EPA by pregnant women in terms of a bleeding tendency during delivery, we have been searching for alternative food functional ingredients to replace EPA. Palmitoleic acid, an n-7 unsaturated fatty acid, has recently been suggested to improve type 2 diabetes and to have anti-inflammatory effects. It has been called a lipokine because of its versatile physiological functions [57, 58]. Here, we describe the physiological properties of palmitoleic acid as a functional lipid that has been elucidated to date.

Among the unsaturated fatty acids, palmitoleic acid (C16:1, n-7; also known as 9-hexadecenoic acid) is a fatty acid found in blood and tissues, particularly in the adipose tissue and liver [59], as well as in human milk [60]. Palmitoleic acid is an n-7 monounsaturated fatty acid that is also naturally present in plants and fish oils [61, 62]. The classification of unsaturated fatty acids and the position of n-7 monounsaturated fatty acids are shown in Figures 2 and 3. In the human body, palmitoleic acid exists as cis and trans isomers. Cis-palmitoleic acid (cis-C16:1n-7, CPA) is synthesized from palmitic acid (C16:0), a saturated fatty acid produced in the body, in addition to normal dietary intake), which is synthesized mainly in the liver by stearoyl-CoA desaturase 1 (SCD1) (Δ9 desaturase) and is incorporated into triglycerides and other products. A portion is further converted to cis-vaccenic acid (cis-C18:1n-7) by an elongase-mediated elongation reaction [63]. Trans-palmitoleic acid (trans-C16:1n-7, TPA) is not biosynthesized from saturated fatty acids in the body and is mainly derived from trans fatty acids found in dairy and in ruminants [64, 65]. Ruminant trans-vaccenic acid (trans-C18:1n-7) is also biosynthesized endogenously in the human body by a chain-shortening reaction [66]. The metabolism of CPA and TPA as palmitoleic acid in the body is shown in Figure 2. Fish oil and macadamia nut oil are dietary sources with higher amounts of CPA than other vegetable oils [67, 68, 69]. In addition, the oil of the fruit of sea buckthorn (Hippophae rhamnoides L.), which is widely grown in Asia, Europe, and Canada, contains about 40% palmitoleic acid [70, 71]. TPA is found in full-fat dairy products, and plasma concentrations of TPA correlate with the intake of full-fat dairy products and butter [72, 73]. Presumably, TPA is typically obtained by ingesting full-fat dairy products. Palmitoleic acid derived from food and its metabolism in the body are shown in Figure 4.

Figure 2.

Classification of fatty acids. Fatty acids are broadly classified into unsaturated and saturated fatty acids, depending on whether the molecule contains or does not contain double bonds between the carbons. Saturated fatty acids are abundant in meat and milk and include palmitic acid (16:0) and stearic acid (18:0). Unsaturated fatty acids are further classified into monounsaturated fatty acids, which have one double bond, and polyunsaturated fatty acids, which contain two or more double bonds. Among monounsaturated fats, oleic acid (18:1), found in olive oil, which has one double bond on the ninth carbon in the chain, is termed an n-9 unsaturated fatty acid. Palmitoleic acid (16:1), a fatty acid with one double bond on the seventh carbon, is referred to as an n-7 unsaturated fatty acid. Palmitoleic acid is further classified into two structural isomers: cis-type palmitoleic acid (CPA), which is abundant in meat fats and oils and in sea buckthorn fruit oil; and trans-type palmitoleic acid (TPA), which is found in milk and dairy products. Polyunsaturated fatty acids include eicosapentaenoic acid (20:5, EPA) and docosahexaenoic acid (20:6, DHA), which are found in fish oil, with the first double bond found on the third carbon from the methyl terminal of the carbon chain. Linoleic acid (18:2), a polyunsaturated fatty acid abundant in plant oil, has its first double bond on the sixth carbon from the methyl end of the carbon chain.

Figure 3.

Structural formulas for three functional lipids. (1) Eicosapentaenoic acid (EPA), an n-3 unsaturated fatty acid. (2) Cis-palmitoleic acid (CPA), an n-7 unsaturated fatty acid. (3) trans palmitoleic acid (TPA), an n-7 unsaturated fatty acid.

Figure 4.

Palmitoleic acid as a food and its metabolism in the human body. Palmitoleic acid (CPA) and trans-palmitoleic acid (TPA) have different routes of intake and metabolism in the body. In the human body, a double bond is introduced into palmitic acid, which is biosynthesized via de novo fatty acid synthesis in the liver and other organs, by SCD1 to form CPA. TPA cannot be biosynthesized in the human body and can only be obtained from milk and dairy products of ruminant origin. In ruminants, the gut bacteria biosynthesize conjugated linoleic acid, rumenic acid, from linoleic acid and store it as trans-vaccenic acid (TVA) in the body’s adipose tissue, etc. TVA is further converted to TPA in the mammary gland and secreted into ruminant milk. Another route for obtaining TPA is through ingestion of TVA in the fat of pork and beef, which is partially converted to TPA by β-oxidation in the human body.

5. Palmitoleic acid as a functional lipid

In humans, palmitoleic acid is abundant in the muscle, liver, and adipose tissue, although the content of palmitoleic acid in the adipose tissue decreases with age [74, 75]. Cis-palmitoleic acid, an adipose-derived lipid hormone (lipokine), is intrinsically synthesized by SCD-1 in adipocytes through palmitic acid and acts in the liver and skeletal muscle to improve insulin sensitivity and metabolism in animal models [58, 76]. In addition, circulating levels of palmitoleic acid in humans are strongly correlated with insulin action [77]. Interestingly, the peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate DNA transcription, and three types are known: PPARα, PPARγ, and PPARδ. The oral administration of palmitoleic acid has been shown to suppress feeding by increasing cholecystokinin levels without involving this PPARα pathway [78]; moreover, it decreases phosphorylation of NF-κB p65 in mouse liver macrophages and inhibits the expression of inflammatory cytokines [79]. Palmitoleic acid also suppresses gene expression of palmitin-induced inflammatory cytokines via AMP-activated protein kinase, indicating a mechanism by which it exerts an anti-inflammatory effect [80]. Topical administration of palmitoleic acid has been reported to heal wounds due to its anti-inflammatory effects [81]. In humans, an association between dietary-derived and blood palmitoleic acid levels and a decreased incidence of diabetes, risk of cardiovascular disease, and inflammatory status have been suggested [77, 82, 83]. Thus, palmitoleic acid is thought to have an inhibitory effect on the development of diabetes by improving insulin resistance and increasing pancreatic beta cell proliferation and insulin secretion in animal and cellular models [84, 85, 86, 87, 88, 89].

Thus, in summary, many studies have shown that palmitoleic acid, as a functional lipid, is effective in preventing the onset of lifestyle-related diseases and has anti-inflammatory, anti-diabetic, and anti-obesity effects. Palmitoleic acid has two structural isomers: the cis-type CPA and the trans-type TPA. The former is biosynthesized from palmitic acid, a saturated fatty acid that causes inflammation and organ stress in skeletal and cardiac muscle, by introducing double bonds and chain-elongation reactions, while the latter cannot be synthesized in the human body and is derived from dietary fatty acids found in meat and milk of ruminant origin (Figure 4).

Little research has been done on the differential effects of the CPA and TPA isomers on biological functions. Recently, we found that high-glucose-exposed myoblasts show increased phosphorylation levels of MAPK/ERK1/2, ROS production, and inflammatory signals; however, their treatment with TPA and EPA suppressed both ROS production and inflammation. In contrast, the opposite was found with a CPA treatment, which increased ROS production and demonstrated higher cytotoxicity than TPA [90]. Thus, CPA and TPA are functional lipids that may affect the organism differently.

Recently, an increasing number of clinical studies have focused on TPA derived from dairy products. In the Cardiovascular Health Study, a large prospective cohort study in the United States, it was found that older adults with a higher percentage of TPA in their blood total fat content had lower insulin resistance and a significantly lower incidence of type 2 diabetes [91]. The Multi-Ethnic Study of Atherosclerosis (MESA) found that TPA in blood was associated with elevated LDL cholesterol, but reduced triglycerides, fasting insulin, blood pressure, and diabetes incidence [92]. Two cohort studies, the Nurses’ Health Study and the Health Professionals Follow-Up Study, of 3333 adults without diabetes aged 30–75 years, found that higher plasma milk-derived TPA levels were associated with a lower risk of developing diabetes mellitus [93]. In a meta-analysis examining the association of saturated and trans-unsaturated fat intake with all-cause mortality, cardiovascular disease, coronary heart disease, ischemic stroke, and type 2 diabetes, TPA intake from a ruminant-derived diet was inversely associated with type 2 diabetes [94]. Another study showed that, as a potential biomarker of milk fat intake, high levels of TPA in circulating blood or adipose tissue were associated with a lower risk of developing type 2 diabetes [95].

In a nonclinical basic research study using db/db mice, TPA was shown to ameliorate hypercholesterolemia by reducing serum cholesterol, low-density lipoprotein, high-density lipoprotein, and hepatic free cholesterol levels, while CPA had none of these effects [96]. TPA also inhibited cholesterol absorption from the intestinal tract by markedly reducing the expression of intestinal Niemann-Pick C1-like 1 protein. However, histological examination of the liver showed that CPA more effectively ameliorated hepatic lipidosis than did TPA. These results suggest that TPA and CPA may prevent hypercholesterolemia by different mechanisms [96]. In a mouse model of diet-induced obesity, TPA prevented weight gain caused by a high-fat diet, reduced visceral adipose tissue weight and adipocyte size, and increased expression of lipolysis-related genes [97]. In vascular endothelial cells and hepatocytes, TPA reduced TNF-α-induced inflammation [98], and in experiments with β-cell lines, TPA and the branched-chain fatty acid 15-methylhexadecanoic acid (iso 17:0) increased PPARγ activity by about two fold and enhanced β-cell function, thereby improving insulin resistance. TPA has been reported to improve insulin resistance by increasing β-cell function [99]. Palmitic acid and CPA decreased hepatocyte viability at concentrations above 1 mM, whereas TPA treatment had a cell proliferative effect: TPA had a beneficial effect on hepatocyte survival signals by activating sirtuin 1 and inducing PPARα activity [100].

In the Ludwigshafen Risk and Cardiovascular Health (LURIC) clinical study of the association between trans fatty acids and mortality, TPA was associated with a reduced risk of death due to cardiovascular causes, but this effect was not observed with industrially produced hydrogenated fats and oils [101]. The effects of palmitoleic acid were examined using isoproterenol-induced myocardial damage model mice and primary mouse cardiomyocytes, and the results showed that palmitoleic acid may have a protective effect against cardiac fibrosis and inflammation by regulating PPAR-specific signaling pathways in the heart [102].

6. Conclusions

When a woman has diabetes during pregnancy, her hyperglycemia creates a hyperglycemic intrauterine environment for the fetus via the placenta. Exposure to this environment during the fetal period is closely related to the future development of cardiac and neurological diseases in the offspring. We have been conducting research to find food ingredients that reduce insulin resistance in pregnant women. While EPA has anti-diabetic effects, there are concerns about the active use of EPA in pregnant women due to its anticoagulant effect and due to the marine pollution of fish oil, which is a primary source of EPA supplementation. In this paper, we propose the use of palmitoleic acid, an n-7 unsaturated fatty acid, which has anti-obesity, lipid metabolism improving, and cardioprotective effects similar to those of EPA. Palmitoleic acid is classified into cis-type CPA and trans-type TPA. Further basic molecular studies are needed to clarify the differences in the mechanisms of action of these structural isomers. In particular, TPA has beneficial effects on the cardiovascular system. However, the intake of TPA, which is only marginally present in dairy products, must be carefully considered from a mother-child nutritional perspective, as excessive intake of dairy products by the mother may lead to overnutrition and allergic reactions in the child. Recently, a synthesis that converts CPA, which is abundant in macadamia nut oil and sea buckthorn fruit oil, to TPA has been developed, and research on palmitoleic acid-producing bacteria is underway. Thus, it is expected that TPA will be available as an inexpensive, safe, and accessible supplement in the future.

Acknowledgments

We gratefully acknowledge the work of past and present members of our laboratory. This work was supported in part by the JSPS KAKENHI Grants (nos. 22 K 11882, 20 K11611, and 18 K11136 to AN and RK), the Dairy Products Health Science Council and Japan Dairy Association (to AN), and the Research Program of Jissen Women’s University (to AN).

Conflict of interest

The authors declare no conflict of interest.